Microsatellite markers for the fungal banana pathogen Mycosphaerella musicola

Yellow Sigatoka caused by the ascomycete Mycosphaerella musicola Leach, is one of the most severe banana diseases worldwide, which spread in most banana growing areas, until Black Sigatoka, a more aggressive disease caused by Mycosphaerella fijiensis, appeared. Because of the highly devastating nature of the latter pathogen, recent research almost exclusively focused on M. fijiensis. To close the gap of knowledge and to study the population structure of M. musicola in Yellow Sigatoka‐infested areas, we cloned and characterized a versatile set of 26 polymorphic locus‐specific microsatellite markers.     

Evaluation of plant extracts as an antagonist to mycelial growth of Mycosphaerella fijiensis morelet

Three plant extracts (rice husk, wood and bamboo) at different concentration were evaluated in vitro as an antagonist to mycelial growth of Mycosphaerella fijienesis on different culture media using spread plate and mycelia dry weight method. The plant extracts had significant effects on the mycelial growth of Mycosphaerella fijiensis. Rice husk extract at concentration of 1, 1.5, 2.5 and 5% completely inhibited the mycelia growth of M. fijiensis in malt extract broth (MEB) and at 2.5 and 5% on malt extract agar (MEA). Wood extract at concentration of 1 and 1.5% inhibited the mycelial growth of M. fijiensis and completely at concentration of 2.5 and 5% on MEA. Although complete inhibition was only observed at 5% concentration on MEA for bamboo extract, the evaluated plant extracts could be recommended for the control of M. fijiensis on a large-scale farming.     

Development of polymorphic microsatellite markers for the Eucalyptus leaf pathogen Mycosphaerella nubilosa

Mycosphaerella nubilosa is one of the most important Eucalyptus leaf pathogens, causing premature defoliation and stunting of growth. The aim of this study was to develop polymorphic microsatellite markers for M. nubilosa. Fifteen primer sets were developed and evaluated for polymorphism. Two primers were monomorphic, three primers did not amplify the desired region and 10 primer pairs were polymorphic. These microsatellite markers will be applied to population biology studies of M. nubilosa collections from several countries. These studies will promote an understanding of the genetics and the global movement of M. nubilosa that is severely limiting plantation development.     

Molecular Variability of Mycosphaerella graminicola as Detected by RAPD Markers

A total of 90 isolates of Mycosphaerella graminicola, the cause of septoria tritici leaf blotch of wheat, were tested for DNA polymorphism using 15 decamer random primers. There was a high level of genetic variability among isolates. In 131 random amplified polymorphic DNA (RAPD) fragments, which were produced, 96% were polymorphic. Based on multilocus analysis, 40 different molecular phenotypes were detected. These molecular phenotypes were randomly distributed among sampling sites, suggesting that no clonal structure existed in the population. Cluster analysis showed that the maximum similarity value among isolates was approximately 81% and no identical isolates were detected, indicating that every isolate was a unique genotype. The high degree of DNA polymorphism, the large number of different molecular phenotypes, their random distribution and the results of the cluster analysis all suggested that sexual reproduction has a major role in the genetic structure of M. graminicola in western Canada. The presence of sexual reproduction provides the opportunity for development of new virulent genotypes in the population and suggests that the pathogen may adapt rapidly to any race‐specific sources of resistance. Therefore, when breeding for resistance to M. graminicola, emphasis should be placed on use of non‐race‐specific resistance.     

Polymerase chain reaction–restriction fragment length polymorphism markers for the fungal banana pathogen Mycosphaerella fijiensis

Polymerase chain reaction–restriction fragment length polymorphism markers were developed to study populations of the fungal banana pathogen Mycosphaerella fijiensis. Twelve markers were defined, 11 in anonymous and single‐copy nuclear DNA sequences and one in the internal transcribed spacer and 5.8S rDNA sequence. The polymerase chain reaction products obtained with locus‐specific primer pairs were digested with restriction enzymes to reveal polymorphism. Between five and 12 markers were polymorph in M. fijiensis populations from different geographical origins (Papua New Guinea, the Philippines, Cameroon and Latin America). The mean of allele number and gene diversity (expected heterozygosity) per locus in the different geographical populations ranged between 1.4 and 2.7 and 0.17 and 0.45, respectively.     

Antifungal activity of novel indole derivative from endophytic bacteria Pantoea ananatis 4G-9 against Mycosphaerella musicola

An endophytic bacterium isolated from banana G-9 (AAA genotype) leaves exhibited strong antagonistic activity against Mycosphaerella musicola. The isolate was identified as Pantoea ananatis 4G-9 by 16S rRNA sequence analysis. Secondary metabolite obtained from P. ananatis 4G-9 was found to have antifungal activity. The active compound was purified from crude extract using column chromatography. Purity of the active compound was assessed using high-performance liquid chromatography. Spectral analysis of compound using infrared, mass spectrometry and nuclear magnetic resonance indicated that the compound structure is an indole derivative. The compound showed strong and dose-dependent antifungal activity against M. musicola. This is the first report on P. ananatis isolated as an endophyte from banana leaves and its antifungal activity against M. musicola.     

Oligonucleotide Fingerprinting Detects Genetic Variability at Different Levels in Nigerian Mycosphaerella fijiensis

DNA fingerprinting with synthetic simple repetitive oligonucleotides such as (CA)₈ or (CAA)₅ detected polymorphisms between various isolates of the ascomycete Mycosphaerella fijiensis, the causal agent of the black Sigatoka disease of Musa. These microsatellite motifs are present at multiple chromosomal locations and in high copy numbers in the Mycosphaerella genome, generating informative fingerprints with low background. Variability exists on a macro- as well as a microgeographical scale: it occurred within one lesion, between lesions of one plant, between plants, cultivars, and geographic locations. Mathematical analysis of the data produced dendrograms that demonstrated the presence of different genetically related groups of Mycosphaerella fijiensis in Nigeria.     

Selection ofMycosphaerella fijiensis-resistant cell lines from micro-cross sections of banana and plantain

Anin vitro selection system using microcross sections of banana and plantain cultivars belonging to AAA and AAB genomic groups were used to produce plants resistant against the Black Sigatoka disease. The fungus resistant plantlets were obtained in a double selection system. This involved in a first step the use of a fungal crude filtrate and in the second step the purified host-specific toxin 2,4,8-trihydroxytetralone extracted from the fungusMycosphaerella fijiensis (M. fijiensis), the causal agent of Black Sigatoka disease. Resistant plantlets obtained from the double selection system were inoculated with conidia ofM. fijiensis in a growth chamber to reproduce Black Sigatoka symptoms. Compared to non-treated control plantlets, which were highly susceptible to the fungus, 10.7–19.3% toxin-resistant plantlets which arose from tissues that went through the double selection system were resistant againstM. fijiensis. This technique of using micro-cross sections for selection on fungal toxins seems to be amenable to differentMusa genotypes for the production of fungus-resistant plants.F. A. Schulz died 11. 3. 1995     

Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation

The Fertile Crescent represents the center of origin and earliest known place of domestication for many cereal crops. During the transition from wild grasses to domesticated cereals, many host-specialized pathogen species are thought to have emerged. A sister population of the wheat-adapted pathogen Mycosphaerella graminicola was identified on wild grasses collected in northwest Iran. Isolates of this wild grass pathogen from 5 locations in Iran were compared with 123 M. graminicola isolates from the Middle East, Europe, and North America. DNA sequencing revealed a close phylogenetic relationship between the pathogen populations. To reconstruct the evolutionary history of M. graminicola, we sequenced 6 nuclear loci encompassing 464 polymorphic sites. Coalescence analyses indicated a relatively recent origin of M. graminicola, coinciding with the known domestication of wheat in the Fertile Crescent around 8,000-9,000 BC. The sympatric divergence of populations was accompanied by strong genetic differentiation. At the present time, no genetic exchange occurs between pathogen populations on wheat and wild grasses although we found evidence that gene flow may have occurred since genetic differentiation of the populations.     

Genetic structure of the global population of banana black leaf streak fungus, Mycosphaerella fijiensis

The genetic structure of Mycosphaerella fijiensis populations around the world was examined using DNA restriction fragment length polymorphism (RFLP) markers. Allele frequencies at 19 nuclear RFLP loci were estimated in a sample of 136 M. fijiensis isolates from five geographical populations representative of banana-producing areas (South-East Asia including the Philippines and Papua New Guinea, Africa, Latin America and Pacific Islands). Within each population, gametic disequilibrium tests between the 19 nuclear RFLP loci were mainly non significant ( P > 0.05), indicating that random sexual reproduction occurred in these populations. All M. fijiensis populations had a high level of genotypic and allelic diversity ( H , gene diversity: 0.25–0.59). The highest levels of gene diversity were found in the two South-East Asian populations ( H : 0.57 and 0.59). Most of the alleles (> 88%) detected in Africa, Latin America and Pacific Islands populations were also detected in South-East Asian populations. Furthermore, a high and significant ( P < 0.05) level of genetic differentiation was observed among M. fijiensis geographical populations (overall estimate of F_st : 0.32). These results were consistent with the hypothesis that M. fijiensis originated in South-East Asia and spread recently to other parts of the world. The level of allelic diversity in M. fijiensis populations from regions other than South-East Asia was drastically reduced, indicating founder effects. The data also suggested rare occurrence of migration of M. fijiensis between continents.     

Genetic discontinuities and disequilibria in recently established populations of the plant pathogenic fungus Mycosphaerella fijiensis

Dispersal processes of fungal plant pathogens can be inferred from analysis of spatial genetic structures resulting from recent range expansion. The relative importance of long‐distance dispersal (LDD) events vs. gradual dispersal in shaping population structures depends on the geographical scale considered. The fungus Mycosphaerella fijiensis, pathogenic on banana, is an example of a recent worldwide epidemic. Founder effects in this species were detected at both global and continental scale, suggesting stochastic spread of the disease through LDD events. In this study, we analysed the structure of M. fijiensis populations in two recently (∼1979–1980) colonized areas in Costa Rica and Cameroon. Isolates collected in 10–15 sites distributed along a ∼250‐ to 300‐ km‐long transect in each country were analysed using 19 microsatellite markers. We detected low‐to‐moderate genetic differentiation among populations in both countries and isolation by distance in Cameroon. Combined with historical data, these observations suggest continuous range expansion at the scale of banana‐production area through gradual dispersal of spores. However, both countries displayed specific additional signatures of colonization: a sharp discontinuity in gene frequencies was observed along the Cameroon transect, while the Costa Rican populations seemed not yet to have reached genetic equilibrium. These differences in the genetic characteristics of M. fijiensis populations in two recently colonized areas are discussed in the light of historical data on disease spread and ecological data on landscape features.     

Reactive Oxygen Species and Cellular Interactions Between Mycosphaerella fijiensis and Banana

Globally, the banana plant (Musa spp) is the fourth most important crop after rice, wheat and corn (based on production in tons). It is cultivated in more than 100 tropical and subtropical countries, mainly by small producers and is a fundamental food source for millions of people. Black leaf streak disease (BLSD), caused by Mycosphaerella fijiensis Morelet (sexual phase) or Paracercospora fijiensis (Morelet) Deighton (asexual phase), is the main disease affecting the world??s banana culture. This disease has a wide geographical distribution accounting for losses exceeding 50% of global banana production. We conducted a comparative histocytological study on the kinetics of the infection process using three banana genotypes with phenotypes that differ in resistance to BLSD: Grand Naine (Susceptible), Pisang Madu (Partially Resistant) and Calcutta 4 (Resistant). Experiments were conducted under controlled conditions with the objective of characterizing the cellular interaction processes between M. fijiensis and Musa acuminata. Conidia germination occurred 24 hours after inoculation. Germination rates were high (97%) and there were no significant differences between the three genotypes (P?>?0.147). The Peroxidase enzyme and H₂O₂ were associated with a hypersensitivity-like reaction in the resistant genotype Calcutta 4, indicating a possible role of the enzyme or its product as defense mechanisms against M. fijiensis in banana plants.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

In vitro evaluation of Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet)

In this work, 90 dichloromethane and methanol extracts obtained from 45 plants collected at the Natural Reserve Bremen – La Popa (Colombia) and at the Natural Regional Park Ucumarí (NRPU, Colombia) belonging to five botanical families were evaluated at 1000 mg/l, for their in vitro fungicide activity through the ascospore germ tube elongation and the measurement of the mycelial radial growth of Mycosphaerella fijiensis assays. The methanol extracts from the species Lycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as, the dichloromethane extracts from P. pesaresanum and those from the Lauraceae family named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensis ascospore germination in 100% in the germinative tube elongation assay. With regards to the effects of the plant extracts on mycelial radial growth, the methanol extracts from P. pesaresanum and the dichloromethane one from N. acutifolia both showed 100% inhibition in this bioassay. Additionally, from the phytochemical screening on the dichloromethane and methanol extracts it was found that compounds such as alkaloids, phenols and terpenes were present in most of the extracts evaluated and they might be the cause of the antifungal activities reported.     

Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.     

Phenylphenalenones protect banana plants from infection by Mycosphaerella fijiensis and are deactivated by metabolic conversion

Phenylphenalenones, polycyclic aromatic natural products from some monocotyledonous plants, are known as phytoalexins in banana (Musa spp.). In this study, ¹H nuclear magnetic resonance (NMR)‐based metabolomics along with liquid chromatography and mass spectrometry were used to explore the chemical responses of the susceptible ‘Williams’ and the resistant ‘Khai Thong Ruang’ Musa varieties to the ascomycete fungus Mycosphaerella fijiensis, the agent of the black leaf Sigatoka disease. Principal component analysis discriminated strongly between infected and non‐infected plant tissue, mainly because of specialized metabolism induced in response to the fungus. Phenylphenalenones are among the major induced compounds, and the resistance level of the plants was correlated with the progress of the disease. However, a virulent strain of M. fijiensis was able to overcome plant resistance by converting phenylphenalenones to sulfate conjugates. Here, we report the first metabolic detoxification of fungitoxic phenylphenalenones to evade the chemical defence of Musa plants.     

Potential use of Trichoderma-based bioproduct for black leaf streak disease (Mycosphaerella fijiensis) management in the field

Organic banana production demands alternatives for controlling black leaf streak disease caused by Mycosphaerella fijiensis Morelet. The foliar application of a Trichoderma-based bioproduct increased the plant growth and provides some degree of control by reduction of the disease severity in two growing cycles of the banana plants cv. ‘Williams’.     

Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis,causal agent of the black leaf streak disease of banana

Previously, we have determined the nonhost‐mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf‐4 and Cf‐Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2‐2 and MfEcp2‐3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf‐4 and Cf‐Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co‐evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South‐East Asia relative to strains from other banana‐producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian‐Pacific region. Furthermore, transient co‐expression of the MfAvr4 effector alleles and the tomato Cf‐4 resistance gene, as well as of MfEcp2, MfEcp2‐2 and MfEcp2‐3 and the putative Cf‐Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field.